Arsenosobenzene pesticide



2,967,799 v ARSENOSOBENZENE PESTICIDE Donald C. Wehner, Stamford, Conn., assignor to American Cyanarnid Company, New York, N.Y., a corporation of Maine No Drawing. Filed May 21, 1959, Ser. No. 814,686

9 Claims. (Cl. 167-30) This invention relates to a method for controlling the growth and development of thallophytes in industrial substrates; i.e., in areas and on materials where the growth of these fungi, bacteria, algae or other vegetable organisms is undesirable. The principal object of the invention is the provision of a broad-spectrum material that can be used as a controlling agent for a wide variety of purposes. I have found that arsenosobenzene is such an agent. The process of my invention, in its broadest aspects, therefore consists essentially in the steps of incorporating arsenosobenzene or its equivalent into a substrate to be protected against the growth of thallophytes.

Arsenosobenzene is a tervalent arsenic compound having the formula I -As= It was first investigated as an anti-syphilitic drug during the classical researches of Ehrlich and has since been evaluated against trypanosomes and other protozoa by Eagle and his associates at Johns Hopkins Hospital but is much too toxic for administration to humans or animals. that it is an extremely effective fungicide andbactericide and that it will inhibit the growth of thes'e'an'd "other vegetable microorganisms in a wide variety of media and at high dilutions. This discovery, plus .thefact that it is a compound of low molecularweight that is easily prepared, and the further fact that it is soluble in alcohols and aqueous alkalies, makes it an important industrial agent for the purpose indicated. 7

As used in the present-specification and claims the term substrate has its usual biochemical meaning of a passive or inert substance acted upon by a microorganism and includes solid, liquid and plastic media but excludes living animals. I have found that by incorporating even small amounts of arsenosobenzene into such substrates the growth of thallophytes therein or thereon is effectively prevented, and the amounts which accomplish this purpose are sometimes hereinafter referred to as growthinhibiting amounts. In general, the quantity of arsenosobenzene that will kill or inhibit the growth of a thallo phyte in an aqueous or other liquid medium providing maximum contact is on the order of from 0.5 to about 50 parts per million. However in other substrates such as solidified paint films, for example, there is only a limited contact between the arsenosobenzene and the microorganism and therefore higher concentrations of arsenosobenzene up to as much as 0.5% on the weight of the substrate may be advisable. Optimum quantities can usually be determined by suitable preliminary tests, and therefore the invention is not dependent upon the ratio of arsenosobenzene to substrate.

As is indicated above, a wide variety of industrial substrates are subject to attack by thallophytes and can therefore advantageously be treated with arsenosobenzene in accordance with the princip'es of my invention. Industrial liquids such as paper mill feed water, paper mill white *f-ttes Patent My present invention is based on the discovery water systems, oil well flood waters, industrial emulsions such as cutting oils, water base paints, latex emulsions and the like and solid materials such as paper, textiles, leather, wood, molded plastics and the like are illustrative of the wide variety of substrates that may be treated. Typical procedures for the application of arsenosobenzene to these substrates, and the results obtained, are described in detail in the following examples.

The invention will be further described and illustrated by these examples to which, however, it is not limited. On the contrary, it will be understood that a wide variety of other substrates can be treated by the described procedures, and that variations and substitutions of equivalents may be resorted to within the customary practices of those skilled in the art.

EXAMPLE 1 Protection of cellulosic textiles Cellulosic textile materials such as cotton cloth, jute burlap and twine,'hemp rope and the like can be protected against mildew by applying arsenosobenzene thereto. This can be shown by the following test procedures.

Suitable aliquots of aqueous arsenosobenzene solution are mixed in sterile Petri dishes with an agar medium containing peptone and the usual mineral salts. After the agar has hardened the surface is inoculated .with a test fungus known to cause mi dew on cellulosic materials and incubated at 25 30 C. for at least two days. A growth-inhibiting concentration of the fungicide is shown when the plate shows no growth after this time.

The fungi listed in Table l are representative of those responsible for the formation of mildew on cellulosic textiles. The minimum concentration of arsenosobenzene capable of inhibiting growth under the test conditions described is indicated in this table in parts per million.

TABLE 1 Fungus Arsenosobeuzene,

p.p.m.

' Cotton cloth such as cotton poplin or denim or heavier cloth such as canvas can therefore be made mildew proof by applying arsenosobenzene thereto. One convenient method of application consists in moistening the cloth by padding or spraying with a water solution of an alkali metal phenyl arsenite obtained by dissolving arsenosobenzene in an aqueous solution of an alkali metal hydroxide such as sodium hydroxide and then wetting it with a dilute aqueous acid such as sulfuric or phosphoric acid. Quantities of about 0.001% to 0.5% of arsenosobenzene, based on the weight of the cloth. can easily be applied by this procedure and will effectively protect the cloth against fungus attack. Other cellulosic articles such as rope, twine, nets, heavy paper such as wrapping paper, camouflage paper and the like can be protected by the same procedure, preferably by applying the phenyl arsenite solution as a spray. On these substrates the step of neutralizing with an acid can be omitted if desired since the alkali metal phenyl arsenites are equally good fungicides.

EXAMPLE 2 Protection of leather Fungi of the classes represented in Table 2 are known to cause mildew on leather. The effectiveness of arsenosobenzene in inhibiting their growth in agar cultures was tested by the procedure described in Example 1. The

minimum growth-inhibiting concentrations, expressed as parts per million, were as follows:

Leather can therefore be protected against these and other mildew-causing organisms by impregnating it preferably uniformly with from about 0.05% to 0.5% or more of arsenosobenzene. The leather may be sprayed: or .otherwise impregnated with water solutions of sodium .phenyl arsenite or the arsenosobenzenemay be applied as a solution in an organic solvent such as ethanol or ethylene glycol.

EXAMPLE 3 Protection of flooding water Water floodingis used in oil fields to achieve secondary "recovery of petroleum and also to repressurize formations and reverse the settling of .land areas. Ordinarily it involves pumping large quantities of water down several wells drilled into an oil-producing formation .and known as injection wells. The water flows through theformation toward oneor more producing wells and carries the petroleum oil with it.

Sulfate-reducing bacteria, .the most common of which 'is"Desulf0.vibrio desulfricans, are almost always .present in the flood water. These are anaerobes and cannot grow in the presence of atmospheric oxygen, but in the absence or molecular oxygen they reduce sulfates to hydrogen sulfide. They also .cause extensive corrosion of. iron and steel by oxidizing cathodic hydrogen. Treatment of the flood water with a bactericide is therefore necessary.

One approved method for evaluating the bactericidal efiiciency of a compound for use in injection Water is the A'.P.I. bacteriostatic test procedure. A sulfate reducer medium for this test is as follows:

Added after sterilization.

.All the ingredients except the iron compound are dissolved in the water by gentle heating with-constant stirring and the pH is adjustedto 7.5 .with NaOH. If .necessary themediumis then filtered. It is autoclaved at lbs. steam pressure for 10 minutes, cooled slightly and the iron salt is added. After cooling, the' medium .is inoculated by adding ten ml. of inoculum to each liter of medium. The inoculum should be from the' third successive 24 hour transfer .of an actively growing culture of the. sulfate reducing bacteria.

Mid-Continent, Strain .A-Texas Company has .been .fdesignated by the. A.P.I. as the test culture of sulfate reducing bacteria .to be usedfor comparativescreening of chemicals. This culture is available-from'the University of .Texas, Department of Bacteriology, at Austin, "Texas. It can be carried on the above-described medium after adding 0.3% of agar. When arsenosobenzene is tested as the bactericide it is preferably first dissolved in alcohol to facilitate measuring. Sufiicient of the solution is added toeach of. several 10ml. clear bottlesso'that when thesecont-ainers are filled, thedesired amountof-arsenosobenzene is present.

4 medium, capped with polyethylene caps and incubated at 35 C.:2 C.

All work is done in duplicate with at least three con trols for each series, the controls consisting of bottles filled with inoculated medium only. Growth of sulfate reducers in the bottles is indicated by an intense blackening of the medium while containers having sufiicient arsenosobenzene to inhibit growth will remain clear. Bottles with no sulfate reducer growth are observed for three Weeks after'the controls have blackened.

By the use of this test procedure arsenosobenzene was found to be an effective bacteriostatic agent against the indicated strain ofsDesulfo'vibrio desulfricans at concentrations of less than 1.5 parts per million. The practical importance of this discovery is evident from the fact that the usual dosage rates of presently used bactericides are from 5 to parts per million in flood water. The quantity of such water in a single flooding operation frequently averages as much as from 100,000 to 500,000 barrels per .day,'and the savings in the quantity of bactericide are therefore very substantial.

.iT-he bottlesare then completely filled=with:the inoculated EXAMPLE 4 In the drilling of oil wells and other deep wells by the rotary drilling. process an aqueous clay suspension known as drilling mud is recirculated through the well. The mud is pumped down through the hollow drill stem and over the drill bit and rises through the well, carrying the cuttings to the surface. As the drilling proceeds to lower levels steel pipe. is cemented into the upper part of the well as a casing.

A number of problems are encountered that can be solved by incorporating arsenosobenzene into such drilling muds. The growth of anaerobic sulfate reducing bacteria such as Desulfovibrio desulfricans in mud lodged between the casing and the wall of the well is a frequent source of corrosion. Decomposition of starch, quebracho, cellulose compounds and other organic drilling mud additives by such molds as the Aspergilli and Penicillinsyas wellas by bacterial attack leads to rapid deterioration of the mud. As is shown in other examples, arsenosobenzenein concentrations of 0.5 to 5 parts per million or greater will check the growth of all of these organisms, and therefore its addition to drilling muds is indicated. Although it is effective at the extremely high dilutions mentioned it is advisable to incorporate larger quantities on the order of 0.05 to 0.5 by weight of arsenosobenzene into the mud'as a safetyfactor. As such muds'are regularly made alkaline by the addition of caustic soda the arsenosobenzene is readily dissolved.

. EXAMPLE 5 One important advantage obtained by using arsenosobenzene in' flood water is that it is also effective against slime-forming bacteria. When added to test tubes containing a peptone dextrose liquid medium inoculated with Bacillus mycoides, with Aerobacter aerogenes and with Pseudomonas aeroginosa and incubated for 18 to '24 'hours it halted the growth of these organisms at concentrations ofless than 0.75 part per million.

This is the test recommended for the evaluation of toxicants for use in slime control by the Biological Con- "trol Committee of the American Paper and Pulp Associ- 'ation; see Microbiology of Pulp and Paper (T.A.P.P.I., 1955), pages 122-123. It will therefore be evident that arsenosobenzene is also an effective bactericide and slime control agent in paper mill white water systems, and may be applied as described in that publication.

. Fungi such 'as the Aspergilli including particularly Asp rgillus niger and certain species of Rhynchosphaeria are capable of utilizing cellulose and cellulose compounds .as sources of "carbon'and are therefore detrimental to paper pulps .and' particularly groundwood pulp. The 'growth :of these and other similar organisms can be ;3':hecked;.byincorporatingsmall amounts of arsenosobenzene on the order of 0.001% to 0.5% by weight into the pulps prior to storage thereof.

EXAMPLE 6 Suitable dilutions of arsenosobenzene were mixed with nutrient salt solutions containing 3 grams NH N0 1 gram K HPO 0.25 gram MgSO; and 0.25 gram KCI per liter and the mixtures were inoculated with an actively growing culture of mixed green algae (Chlorophyceae). The mixtures were incubated in sunny areas of a greenhouse for from 2 to 3 weeks and were then examined. Control flasks containing no fungicide showed a typical green algae growth, whereas those which contained 1.6 parts per million or more of arsenosobenzene showed no growth of algae.

EXAMPLE 7 Ordinary paint is essentially a suspension of a pigment such as titanium dioxide, white lead, lithopone and the like in a drying vehicle which is usually a vegetable drying oil such as linseed oil, soya oil or a mixture of such oils. Films'of such paint are attacked by fungi of the Aspergillus, Penicillium and Cladosporium types and by Pulluloria pullulans in warm and humid areas. I have found that the incorporation of small amounts of arsenosobenzene on the order of 0.01% to 0.5% or more into this type of paint will check the growth of such organisms.

Cultures of Aspergillus niger, Penicillium luteum and Trichoderma viride were prepared in an agar medium containing 1% peptone and 0.1% dextrose. Samples of a commercial house paint having a drying oil vehicle were prepared. Arsenosobenzene was added to some of these samples in amounts of 15 mg. of the fungicide in 30 cc. of paint. The paint samples were then applied to paper and the films were allowed to harden. Each film was then inoculated with one of the cultures and incubated seven days at 28-30 C. and 80-90% relative humidity. It was found that growth of all three of the molds was prevented by the arsenosobenzene whereas there was extensive mold growth on the samples containing no fungicide.

Water paints containing casein or other protein, latex paints and oil emulsion paints are also subject to attack by bacteria and fungi. Arsenosobenzene in amounts of about 0.01% to 0.5 will also check the growth of microorganisms in such compositions both during storage in closed containers and as hardened surface coatings exposed to atmosphere. Typical latex paints that can be protected in this manner are those in which a polymerized acrylic or methacrylic acid ester or a polyvinyl acetate or other polymer is emulsified in an aqueous medium as by emulsion polymerization of their monomers. Other classes of water base paints are those in which an oil modified alkyd resin such as a modified phthalic glyceride resin is emulsified in a water solution of a complex emulsifier as described in US. Patent No. 2,889,293. Samples of commercial acrylic and polyvinyl acetate water base paints and the emulsified alkyd resin paint described in Example 3 of the patent were inoculated with the cultures described above after incorporating 0.05% by weight of arsenosobenzene, applying as films to paper and drying. In all cases the growth of the mold was prevented by the arsenosobenzene.

Culture media of the type described above were inoculated with Spicaria violacea, which is another organism known to cause mildew of painted surfaces, after adding suitable aliquots of arsenosobenzene solution and incubated at about 30 C. for two days using the procedure described in Example 1. The fungicide was effective against this mold in concentrations of 2.5 parts per million and more.

Molded plastics such as those composed of or containing polyvinyl chloride, polyvinyl acetate, polyvinylidene chloride, polyethylene, polystyrene and other thermoplastic hydrocarbons as well as thermosetting resins such as urea-formaldehyde and melamine-formaldehyde resins can also be protected against fungal attack by incorporating therein small amounts of arsensobenzene on the order of 0.1% to 1%. For example a polyvinyl chloride containing dioctyl phthalate and dioctyl sebacate as plasticizers was molded after incorporating 0.5% of arsenosobenzene. Pieces of the molded material resisted mold growth when incubated for one week in Petri dishes containin gagar incubated with Aspergillus flavus, with Aspergillus niger and with Trichoderma viride.

EXAMPLE 8 A problem that is similar in some respects to that of emulsion paints is the one presented by industrial cutting oils. These are essentially emulsions of hydrocarbon lubricating oils or of other lubricants in water. The aqueous phase frequently contains a surface-active material to lower its surface tension and improve the ability to penetrate and wet the surface and may also contain borax or sodium nitrite as a rust inhibitor. The emulsifying agent is usually an alkali metal salt or soap of a petroleum sulfonate, of a higher fatty acid or of rosin or of a mixture such as tall oil. A typical cutting oil may contain 9% of a hydrocarbon oil of low viscosity emulsified in a solution of 1%.sodium petroleum sulfonate, 0.3% sodium naphthenate and 0.1% ethanol in 89.6% of water.

During use these oils become a breeding ground for a wide variety of bacteria and fungi. Among others they have been found to contain Pseudomonas aeruginosa and other species of Pseudomonas, Aerobacter aerogenes, E. coli and a wide variety of yeasts and molds. Investigations have shown that infected oils are a ready means of transferring bacterial infection from one workman to another.

Because the microorganisms grow in the aqueous phase of the emulsions a satisfactory bactericide must be watersoluble but should not interfere with the stability of the emulsion. Arsenosobenzene is an effective sterilizing agent for all such emulsions, but it is preferably first converted into sodium phenyl arsenite by dissolving in aqueous sodium hydroxide. The resulting solution may be mixed with the emulsion without altering its stability or other desirable properties. Quantities corresponding to 0.01% to 0.5% of arsenosobenzene on the weight of the emulsion are recommended, but because of the wide variety in the compositions and fields of use of such cutting oils it is preferable to conduct tests to determine the optimum amount.

EXAMPLE 9 Wood rot is caused by filamentous fungi the most common of which are Lentinus lipideus, Lenitzes trabea and Poria monticola.

Arsenosobenzene was found to be effective in preventing the growth of the first two of these organisms on agar at concentrations of less than 2.5 parts per million when tested by the procedure of Example 1. It was effective against the third when added to a culture of the fungus in the mineral salts solution of Example 5 at the same concentration. It is evident, therefore, that peeled logs such as telephone poles, fence posts and the like as well as shaped or semi-shaped lumber such as railroad ties, building lumber and the like can be protected against attack by these and similar fungi by impregnation with arsenosobenzene. This can be accomplished by spraying or otherwise impregnating the lumber with a solution of the fungicide in ethanol, isopropanol or other volatile solvent.

The development of mildew in the paint and soft rot of the wood in the wet parts of cooling towers is a typical example of an industrial problem requiring a broad-spectrum fungicide. Such fungi as the Cytosporella, Nematogonium, Caniothyrium, Chaetomium and Phialophora have been found in the cooling towers of air conditioning and other systems wherein warm air is contacted with water sprays.

I have found that this problem can be solved by maintaining lo'w "concentrations of arseno'sobenzene in the Ispraywater. In most cases concentrations within the range of about two to six parts per million are sufficient. It will be understood that in most installations of this type the spray water is collected in a sump and recirculated through the sprays'many times so the cost of the fungicide can be held to a minimum.

What I claim is:

1. A method of preventing the growth and development of a microorganism in contact with a substrate substance which is subject to attack by said microorganism and where the growth of the microorganism is undesirable, said microorganism being selected from the [group consisting of bacteria, fungi and algae, which comprises incorporating with said substrate substance arsenosobenzene in growth-inhibiting amounts.

2. A method according to claim -1 wherein the growth 'of these microorganisms in water-spray cooling towers is prevented by dissolving growth-inhibiting amounts of arsenosobenzene in the water circulated in these towers.

3. A method according to claim 1 in which the substrate is a cellulosic material.

4. A method according to claim-3 in which the substrate is a textile.

5."A method according to claim 1 in which the substrate is a nutrient-containing water. a

References 'Cited'in the file ofthis patent UNITED STATES PATENTS 2,280,477 Carter.- Apr; 21, 1942 2,284,889 Fisher June 2, 1942 2,434,291 Smith .Q. Jan. 13, 1948 2,676,147 Mulligan' Apr. 20, 1954 2,686,119 Buckman' Aug. 10, 1954 2,800,425 Smith July 23, 1957 2,867,563 Musser -2--. Jan. 6, 1959 OTHER'REFERENCES U.S. Dispensatory, 2 4th Ed. (1947)," pp.- 107-111.

' Frear:,A Catalogue of Insecticides and-Fungicides,-

1948, vol. 2, p. 41. 

1. A METHOD OF PREVENTING THE GROWTH AND DEVELOPMENT OF A MICROORGANISM IN CONTACT WITH A SUBSTRATE SUBSTANCE WHICH IS SUBJECT TO ATTACK BY SAID MICROORGANISM AND WHERE THE GROWTH OF THE MICROORGANISM IS UNDESIRABLE, SAID MICROORGANISM BEING SELECTED FROM THE GROUP CONSISTING OF BACTERIA, FUNGI AND ALGAE, WHICH COMPRISES INCORPORATING WITH SAID SUBSTRATE SUBSTANCE ARSENOSOBENZENE IN GROWTH-INHIBITING AMOUNTS. 